Absorbing organic reagents into diagnostic test devices by formation of amine salt complexes

ABSTRACT

A method of applying an organic acid reagent having limited transolubility in organic solvents, aqueous solvents, and mixtures thereof to a component of a diagnostic test device by mixing an organic acid reagent and an amine to form a salt complex, dissolving the salt complex in a solvent to release the organic acid reagent into the solvent, and applying the solvent to the component of the diagnostic test device such that the organic acid reagent present in the solvent becomes integrated into the diagnostic test device. The amine is represented by the formula H m NR n  wherein m is 0, 1, or 2; n is 1, 2, or 3; the sum of m and n is 3; and R is an independently selected group which renders the salt complex soluble in aqueous solvents, organic solvents, or mixtures thereof. The amine is also represented by the formula NR 4   +  wherein R is an independently selected group which renders the salt complex soluble in aqueous solvents, organic solvents, or mixtures thereof. The organic acid reagent is selected from a carboxylic acid, a sulfonic acid, a phosphoric acid, and mixtures thereof. Preferred reagents are dyes like pyragallol red. In a specific embodiment the diagnostic test device is a paper test strip.

FIELD OF THE INVENTION

The invention relates to methods for absorbing an organic reagent into acomponent of a diagnostic test device and diagnostic test devicesprepared by such methods. In particular, the invention relates tomethods for absorbing an organic reagent into the absorbent material ofa diagnostic test strip and diagnostic test strips prepared by suchmethods.

BACKGROUND OF THE INVENTION

The use of diagnostic test devices such as dry devices to analyzecomponents in a sample of human body fluid such as urine is well known.A diagnostic test strip is one type of dry device that may be used toanalyze components in human body fluid. Diagnostic test strips aretypically composed of one or more pads of paper attached to a plasticcarrier which serves as a handle.

With diagnostic test strips, a reagent such as an organic molecule isapplied to or absorbed into an absorbent material such as paper bydipping the absorbent material into one or more solvent systemscontaining one or more reagents. Upon dipping the absorbent materialinto the solvent system, the reagent contained in the solvent systembecomes absorbed within or integrated into the fibers of the absorbentmaterial. Once the reagent has been applied to the absorbent material,the absorbent material is dried and assembled into a test strip.

The dried test strip containing the reagent can then be used to test forthe presence of an analyte(s) in test samples. Test samples aretypically human urine or other biological fluids. Test strips arefrequently used to detect proteins in protein assays also referred to astotal protein tests or proteinuria tests or to test for the presence ofparticular proteins in conditions such as albuminuria. Examples ofanalytes which are tested include, but are not limited to, proteins,hormones, drugs, metabolites, glucose, protons (i.e., for pH), ions(i.e., specific gravity), and blood cells. To test for the presence ofan analyte(s), the test strip is dipped into the test sample at whichpoint the reagent in the test strip participates in a reaction sequencewith a particular analyte(s) in the test sample. Upon detecting thepresence of a particular analyte(s) in the test sample, the reagent inthe test strip responds with an instrumentally or visually detectablesignal to the user such as a change in color.

In order for a reagent to become applied to the absorbent material of adiagnostic test device such as a test strip, the reagent needs to besoluble in the solvent system used to apply the reagent to the absorbentmaterial. The more soluble the reagent is in this solvent system, themore reagent becomes applied to the absorbent material and the richerthe color obtained on the absorbent material. A high concentration ofreagent in the absorbent material often allows a rich color indication.

In order for a reagent which has been absorbed into the absorbentmaterial of a diagnostic test device such as a test strip to be used todetect an analyte(s) in a test sample, the reagent that is trappedwithin the fibers of the absorbent material typically needs to besufficiently soluble to be available to interact with the analyte(s) inthe test sample. That is, the reagent that has been absorbed into theabsorbent material typically needs to be soluble in the final test stripenvironment (i.e., the test sample) for solution chemistry to take placeand to give an indication that a particular analyte(s) is present in thetest sample. However, high solubility of the reagent in the test samplemay be undesirable in some systems as problems such as reagent migrationbetween multiple pads on the test strip can occur. An optimal systemwould allow assay designers to control the degree of reagent solubilityin the test sample.

The organic reagents which are typically applied to the absorbentmaterial of diagnostic test devices are either soluble in aqueoussolvents with no solubility or only limited solubility in organicsolvents or are soluble in organic solvents with no solubility or onlylimited solubility in aqueous solvents. In other words, many of theseorganic reagents have no transolubility or only limited transolubilityin organic and aqueous solvents. The lack of transolubility of thesereagents in organic and aqueous solvents has made the application ofcertain reagents to diagnostic test devices and the use of thediagnostic test devices containing these reagents difficult.

To illustrate, a solubility problem arises where an organic reagent mustbe applied to the absorbent material of diagnostic test devices using anorganic solvent because the reagent is readily soluble only in organicsolvents, but the reagent must subsequently be used to detect thepresence of proteins in an aqueous test sample (i.e., urine) in whichthe reagent has no solubility or only limited solubility. For example,pyrogallol red is a dye commonly used in diagnostic protein tests thatis soluble in organic solvents such as methanol but has no solubility oronly limited solubility in aqueous solvents such as water or urine. Onechallenge facing assay designers has been to find a way to apply areagent such as pyrogallol red to test strips via an aqueous solvent andto then use the reagent to detect the presence of proteins in an aqueoustest sample in which the reagent has controlled solubility.

One method that has been used to dissolve organic reagents havinglimited transolubility in organic and aqueous solvents has been toincrease the alkalinity of the aqueous solvent. This approach has beenused to dissolve pyrogallol red dye in aqueous solvents and apply thedye via the aqueous solvent to the absorbent material such as the testpaper. Although pyrogallol red may be applied to a diagnostic testdevice such as a test strip via an aqueous solvent using this approach,the strongly alkaline aqueous solvent needed to dissolve the pyrogallolred causes an unacceptable variation in the color of the test paper,rendering the paper unusable in diagnostic tests.

Another method used to dissolve organic reagents having limitedtransolubility in organic and aqueous solvents has been to add watermiscible cosolvents, such as alcohols, to the solvent system. Thisapproach also has shortcomings. The addition of a water misciblecosolvent may increase the solubility of one component in the solventsystem but may simultaneously decrease the solubility of othercomponent(s). In addition, the amount and type of water misciblecosolvent typically requires careful selection, often through a trialand error process, to increase the solubility of the organic reagentwhile maintaining the solubility of the water soluble components.

Because different reagents have varying degrees of solubility in thesolvents used to apply reagents to the absorbent materials of diagnostictest devices such as test papers, it is sometimes necessary to use amultiple-dip or multiple-step application process. For example, amultiple-dip application process may be used where the desired reagentsare reactive with each other or with one or more components contained inone of the solvent systems. Another challenge facing assay designers hasbeen to find a way to control the transolubility of a reagent(s) where amultiple dip application process is involved. For example, in a two-dipapplication process, a reagent may be applied to a test paper using anaqueous solvent as the first dip solution, yet it is desirable that thereagent remain insoluble in the second, organic dip solution to preventleaching of the reagent into the second dip solution that is beingapplied to the test paper.

For the foregoing reasons, there exists a need for a method to applyreagents having limited transolubility in organic solvents, aqueoussolvents, and mixtures thereof to diagnostic test devices such asdiagnostic test strips via organic solvents, aqueous solvents, andmixtures thereof for subsequent use in detecting the presence of ananalyte(s) in test samples such as aqueous test samples. There alsoexists a need for a method to apply reagents having limitedtransolubility in organic solvents, aqueous solvents, and mixturesthereof to diagnostic test devices via organic solvents, aqueoussolvents, and mixtures thereof (a) without adding, or with reduced needto add, cosolvents which affect the solubility of other components inthe solvent and (b) without subjecting the reagent to harsh chemicalconditions to dissolve the reagent in the desired solvent. A need alsoexists for a method to control the transolubility of a reagent(s) wherea multiple dip application process is involved without side effects suchas leaching of the reagent into subsequent dip solutions. There is alsoa need for a method to apply reagents to diagnostic test devices whichallows assay designers to generally control or choose the degree ofreagent solubility in the test sample. There also exists a need fordiagnostic test strips which can be prepared without the above notedshortcomings and with the above noted advantages.

SUMMARY OF THE INVENTION

According to one embodiment, an organic acid reagent having limitedtransolubility in organic solvents, aqueous solvents, and mixturesthereof is applied to a component of a diagnostic test device by mixingan organic acid reagent and an amine to form a salt complex; dissolvingthe salt complex in a solvent to release the organic acid reagent intothe solvent; and applying the solvent to the component of the diagnostictest device such that the organic acid reagent present in the solventbecomes integrated into the diagnostic test device. The amine isrepresented by the formulaH_(m)NR_(n)wherein m is 0, 1, or 2; n is 1, 2, or 3; the sum of m and n is 3; and Ris an independently selected group which renders the salt complexsoluble in aqueous solvents, organic solvents, or mixtures thereof.

According to another embodiment, an organic reagent having limitedtransolubility in organic solvents, aqueous solvents, and mixturesthereof is applied to a component of a diagnostic test device by mixingan organic reagent and an amine to form a salt complex; dissolving thesalt complex in a solvent to release the organic reagent into thesolvent; and applying the solvent to the component of the diagnostictest device such that the organic reagent present in the solvent becomesintegrated into the diagnostic test device. The amine is selected fromtrimethylamine, triethylamine, tributyl amine, trioctylamine,tris(hydroxymethyl)aminomethane, aminoethanol, butylamine, octylamine,triethanolamine, glucamine, a polyethyleneglycolamine, an amino acid,and mixtures thereof.

According to a further embodiment, an organic acid reagent havinglimited transolubility in organic solvents, aqueous solvents, andmixtures thereof is applied to a paper of a diagnostic test strip bymixing an amine and an organic acid reagent to form a salt complex;dissolving the salt complex in a solvent to release the organic acidreagent into the solvent; and applying the solvent to the paper of thediagnostic test strip such that the organic acid reagent present in thesolvent becomes integrated into the paper. The amine is selected fromtrimethylamine, triethylamine, tributyl amine, trioctylamine,tris(hydroxymethyl)aminomethane, aminoethanol, butylamine, octylamine,triethanolamine, glucamine, a polyethyleneglycolamine, an amino acid,and mixtures thereof, and the organic acid reagent is selected from acarboxylic acid, a sulfonic acid, a phosphoric acid, and mixturesthereof.

According to a still further embodiment, an organic acid reagent havinglimited transolubility in organic solvents, aqueous solvents, andmixtures thereof is applied to a component of a diagnostic test deviceby combining an ionized organic acid reagent and a quaternary ammoniumion by an ion exchange process to form a salt complex; dissolving thesalt complex in a solvent to release the organic acid reagent into thesolvent; and applying the solvent to the component of the diagnostictest device such that the organic acid reagent present in the solventbecomes integrated into the diagnostic test device. The ammonium ion isrepresented by the formulaNR₄ ⁺wherein R is an independently selected group which renders the saltcomplex soluble in aqueous solvents, organic solvents, or mixturesthereof.

According to a still further embodiment, a diagnostic test devicecontaining an organic acid reagent having limited transolubility inorganic solvents, aqueous solvents, and mixtures thereof is prepared bymixing an organic acid reagent and an amine to form a salt complex;dissolving the salt complex in a solvent to release the organic acidreagent into the solvent; and applying the solvent to a component of thediagnostic test device such that the organic acid reagent present in thesolvent becomes integrated into the diagnostic test device. The amine isrepresented by the formulaH_(m)NR_(n)wherein m is 0, 1, or 2; n is 1, 2, or 3; the sum of m and n is 3; and Ris an independently selected group which renders the salt complexsoluble in aqueous solvents, organic solvents, or mixtures thereof.

According to a still further embodiment, a diagnostic test devicecontaining an organic acid reagent having limited transolubility inorganic solvents, aqueous solvents, and mixtures thereof is prepared bycombining an ionized organic acid reagent and a quaternary ammonium ionby an ion exchange process to form a salt complex; dissolving the saltcomplex in a solvent to release the organic acid reagent into thesolvent; and applying the solvent to a component of the diagnostic testdevice such that the organic acid reagent present in the solvent becomesintegrated into the diagnostic test device. The ammonium ion isrepresented by the formulaNR₄ ⁺wherein R is an independently selected group which renders the saltcomplex soluble in aqueous solvents, organic solvents, or mixturesthereof.

The above summary of the present invention is not intended to representeach embodiment, or every aspect, of the present invention. This is thepurpose of the detailed description which follows.

DESCRIPTION OF EMBODIMENTS OF THE INVENTION

Embodiments of the invention are, in part, based on the discovery thatreagents having limited transolubility in organic solvents, aqueoussolvents, and mixtures thereof may be applied to an absorbentcomponent(s) of a diagnostic test device such as a diagnostic test stripvia organic solvents, aqueous solvents, and mixtures thereof and maysubsequently be used to detect the presence of an analyte(s) in organictest samples, aqueous test samples, and mixtures thereof. Reagent(s)having limited transolubility may be applied to diagnostic test devicesvia organic solvents, aqueous solvents, and mixtures thereof using themethods described herein (a) without adding, or with reduced need toadd, cosolvents which affect the solubility of other components in thesolvent and (b) without subjecting the reagent to harsh chemicalconditions to dissolve the reagent in the desired solvent. Thetransolubility of a reagent(s) in the final test sample may be generallycontrolled using the methods described herein. In addition, thetransolubility of a reagent(s) may be generally controlled using themethods described herein where a multiple dip application process isused without side effects such as the reagent leaching into subsequentdip solutions.

It has been discovered that by mixing an amine having certain propertieswith an organic acid reagent having no transolubility or only limitedtransolubility in organic solvents, aqueous solvents, and mixturesthereof, a salt complex is formed which can be dissolved in organicsolvents, aqueous solvents, and mixtures thereof. The salt complex canthen be absorbed into one or more components of a diagnostic test devicesuch as an absorbent material via organic solvents, aqueous solvents,and mixtures thereof. By forming a salt complex to absorb the organicreagent into the component(s) of the diagnostic test device, the organicreagent can be applied to the component(s) evenly, homogeneously and ina controlled fashion.

As used herein, the term “transolubility” indicates that the reagent issoluble in more than one of the following three types of solvents:aqueous solvents, non-aqueous solvents such as organic solvents, andmixtures of aqueous and non-aqueous solvents as opposed to being solublein only one or none of these three types of solvents. Transolubility maybe achieved by selecting an appropriate counterion for the reagent ofinterest.

Suitable diagnostic test device formats for use in embodiments of theinvention include, but are not limited to, dry devices such as teststrips, wands, sticks, tubes, chips, channels, wells, cavities, grids,wafers, disks, plates, and cartridges where reagent is applied as driedfilms, layers, spots, or arrays of spots on an absorbent materialcomposing the device or on an absorbent material component within orattached to the dry device. Examples of suitable absorbent materialsinclude, but are not limited to, papers, fibers, fabrics, non-wovenfiber mats, felts, porous membranes, porous ceramics, porous hydrophilicplastics, porous sponges, hygroscopic gels, hygroscopic polymers, andporous or hygroscopic natural materials. Two examples of suitable papersare Ahlstrom 204 available from Ahlstrom Technical Specialties in Mt.Holly Springs, Pa. and Whatman 3MM available from Whatman Inc. in AnnArbor, Mich., both of which are made from cellulose. The methodsdescribed herein also allow reagent solution to be used in a manual orautomated diagnostic assay system as a stable liquid, for example, in areagent container as part of a diagnostic test kit.

Other suitable formats for the diagnostic test devices for use inembodiments of the invention include, but are not limited to, wires,fibers, wands, sticks, tubes, chips, channels, wells, cavities, grids,wafers, disks, plates, chambers, capsules, and cartridges of glass,non-porous ceramics, plastics, silicones, silicon, othersemi-conductors, metals, and coated papers which may use shape orstructure rather than an absorbent material to hold a reagent. It isalso contemplated that the diagnostic test devices may be microscalefluidic devices and microfluidic assay platforms for performing clinicaldiagnostics such as, but not limited to, disks, chips, “labs on disks”,“labs on chips”, “labs on CDs”, microchannels, microlaboratory arrays,and microlaboratory disks. Such formats may supplement and/or be used inplace of the dry device formats described above. For example, ratherthan using the methods described herein to absorb an organic reagentinto absorbent materials such as papers and building test strips withthese papers, it is contemplated that these papers may instead be formedinto small patches which can be installed into chip devices. It is alsocontemplated that the solutions of organic reagents which may be appliedto the absorbent materials as described herein may instead be dried ontoor injected into defined areas of the diagnostic test units.

Where diagnostic test strips are employed, the test strips may be madefrom a variety of materials and are typically made from one or more padsof paper which are cut and attached to a polymeric or plastic carrier toform the diagnostic strip. Pads for test strips may be made from otherwoven, nonwoven, patterned, or cast materials including natural andsynthetic materials which are capable of absorbing fluid such aspolyester, nitrocellulose, ceramics, and glass fiber.

The process described herein involves (a) mixing an amine havinglipophilic properties, hydrophilic properties, or both lipophilic andhydrophilic properties with an organic acid reagent having notransolubility or only limited transolubility in organic solvents,aqueous solvents, and mixtures thereof to form a salt complex; (b)dissolving the salt complex in an organic solvent, an aqueous solvent,or mixtures of one or more organic solvents and one or more aqueoussolvents to release the reagent into the solvent; and (c) applying thesolvent containing the reagent to a component of a diagnostic testdevice so that the reagent present in the solvent becomes integratedinto the diagnostic test device. For example, the solvent containing thereagent may be applied to the diagnostic test device by dipping anabsorbent component or material such as paper into the solvent so thatthe reagent which is dissolved in the solvent becomes applied to theabsorbent material.

The absorbent component or material such as the test paper can be dried,and the absorbent material containing the dried reagent can be assembledinto a diagnostic test device such as a diagnostic test strip. Thediagnostic test device can then be used in detecting the presence of ananalyte(s) in organic solvents, aqueous solvents, and mixtures thereof.When the diagnostic test device is dipped into a test sample accordingthe methods described herein, the reagent becomes dissolved into thetest sample and becomes available to react with the analyte(s) in thetest sample. As an increased amount of reagent may be applied to theabsorbent component or material by forming the salt complex, anincreased amount of reagent is available to become dissolved in the testsample and to detect the presence of an analyte(s) using the methodsdescribed herein. Hence, the instrumentally or visually detectablesignal to the user (i.e., the change in color) is generally enhancedusing the methods described herein and the diagnostic test devicesprepared by the methods described herein.

The organic reagent to be applied to the diagnostic test device isgenerally an organic acid. The organic reagent serves as a source ofanions for creating the salt complex. The amine to be used in promotingthe solubility of the organic reagent serves as a source of positivecounterion(s) which counter the negative charge provided by the anion(s)of the organic reagent. The charges on the anions and cations may becountered such that a salt complex having no net charge may be formed.

The processes and products described herein offer numerous advantages.By using the methods and products described herein, an organic reagentcan be applied to a diagnostic test device via organic solvents, aqueoussolvents, and mixtures thereof where the reagent would normally besoluble in only organic solvents or in only aqueous solvents. Thisprocess improves the ability to absorb organic reagents having notransolubility or only limited transolubility in organic solvents,aqueous solvents, and mixtures thereof into diagnostic test devicesusing organic solvents, aqueous solvents, and mixtures thereof. By usingthe methods and products described herein, an organic reagent whichwould normally be soluble only in organic test samples or only inaqueous test samples may be used to detect the presence of an analyte(s)in organic test samples, aqueous test samples, and mixtures thereof.

A further advantage of the methods and products described herein is thatthe methods and products allow users to generally control the degree ofsolubility of the resulting salt complex in a particular solvent and theavailability of the organic reagent that is applied to an absorbentcomponent or material such as test strip paper to react with testsamples. The amount and the timing of the uptake of a reagent into theabsorbent material can be controlled as well as the release oravailability of the reagent for reaction with analyte(s) in testsamples. By selecting particular mixtures of organic reagents andamines, the salt complex may be designed to be more or less soluble inparticular solvents where so desired. For example, by selecting atri-n-octylamine for salting as compared to triethylamine, a user mayrender an aryl sulfonate reagent more soluble in an organic solvent. Thedegree of solubility of a salt complex in organic solvents, aqueoussolvents, and/or mixtures thereof can be generally controlled byselecting counterions to the reagent of interest which have greater orlesser aqueous or non-aqueous affinity. Those salts with greater aqueousaffinity have increased hydrophilic character. Those salts with greateraffinity for non-aqueous solvents have increased lipophilic character.

A further advantage of the methods and products described herein is thatthe methods and products allow users to generally alter the selectivityof the desired reagent in its interaction with the analyte(s) in thetest sample. For example, where the final protein-indicating reaction isdependent upon interaction of the organic reagent with one or moreproteins, the sites of these interactions have lipophilic andhydrophilic features with differing degrees of affinity for the saltcomplexes formed from the lipophilic or hydrophilic counterions.

Suitable organic reagents for use in embodiments of the invention havestructural characteristics that allow compatible interactions of theacidic reagent with the anion of the amine such that the salt complexthat is formed will be soluble in organic solvents, aqueous solvents,and mixtures thereof. Where a user desires to increase the solubility ofan organic reagent in organic solvents, the user may wish to select areagent having highly aliphatic or aromatic substituent groups withlipophilic character such as alkyl, halogenated alkyl, aryl, or phenylgroups. Where a user desires to increase the solubility of an organicreagent in aqueous solvents, the user may wish to select a reagenthaving functional groups with hydrophilic properties such as—(CH₂)_(n)—OH, —(CH₂)_(n)—C(O)NH₂, —(CH₂)_(n)—SH, or—CH₂CH₂—(OCH₂CH₂)_(n)OH groups where n is zero or a positive integer. Asused herein, the terms “lipophilic” and “hydrophobic” are synonymous andthe terms “lipophobic” are “hydrophilic” are synonymous.

The organic reagents used to form the salt complex are typically organicacids. Suitable organic acids include, but are not limited to,carboxylic, sulfonic, and phosphoric acids having —COOH, —SO₃H, and—OPO₃H functional groups respectively, although it is contemplated thatany organic molecule which is capable of providing an ionizable group ofsufficient acidity to form a salt complex when combined with an aminecontaining a lipophilic group may be used. Examples of suitable organicreagents include dyes such as pyrogallol red and substitutedphenolsulfonephthaleins.

Other suitable dyes include, but are not limited to, acid alizarinviolet N, acid blacks, acid blues, acid oranges, acid greens, acid reds,acid violets, acid yellows, alizarin red S, alizarin violet 3R, alizarinyellow GG, alphazurine A, amaranth, 8-anilino-1-sulfonic acid (ANS),arsenazo dyes, aurintricarboxylic acid, azocarmine B, benzopurpurin,biebrich scarlet, bordeaux R, brilliant black BN, brilliant blue G,brilliant blue R, brilliant sulfaflavine, brilliant yellow,bromochlorophenol blue, bromocresol green, bromocresol purple,bromophenol blue, bromopyrogallol red, bromothymol blue, bromoxylenolblue, chicago sky blue 6B, chlorophenol red, chromotrope dyes,chromoxane cyanine R, chysophenine, cibacron brilliant red 3B-A,cibacron brilliant yellow 3G-P, cibacron blue 3G-A, congo red,cresolphthalein, cresol purple, cresol red, direct red 75, direct red81, dinitrohexabromosulfonephthalein, eosin B, eosin Y, eriochrome blackT, eriochrome blue black 2B, eriochrome red B, erioglaucine, erythrosinB, ethyl orange, evans blue, fast green FCF, fast yellow, flavazin L,fluorescein, fluorescein water soluble, 2-(4-hydroxyphenylazo)benzoicacid (HABA), 8-hydroxyquinoline-5-sulfonic acid, indigo carmine,indigotrisulfonic acid, indocyanine green, lucifer yellow, merbromin,metanil yellow, methyl orange, methyl red, methylthymol blue, mordantoranges, mordant red, mordant yellows, naphthochrome green, naphthol ASBI phosphate, naphthol blue black, naphthol yellow S, new coccine,nickel phthalocyaninetetrasulfonic acid, nitrazine yellow, nitro red,nitrosonaphtholdisulfonic acid, nuclear fast red, orange G, orange II,palatine chrome black 6BN, patent blue VF, phenolphthalein, phenol red,phloxine B, plasmocorinth B, ponceau S, ponceau SS, primulin,pyrocatechol violet, rosolic acid, rose bengal, tartrazine,tetrabromophenol blue, tetrabromophonolphthalein,tetrabromophenolsulfonephthalein, thymol blue, thymolphthalein,thymolphthalein monophosphoric acid, tiron, tropaeolin O, trypan blue,violamine R, xylenol blue, xylidyl blue, and zincon. Mixtures of one ormore compatible organic reagents may be used in the methods and productsdescribed herein. When the organic acid reagent is a dye, it commonlyserves as a source of anions for forming the salt complex.

The anionic structures of some of the suitable dyes for use inembodiments of the invention are shown as Structures A-E below. Thefirst ionization form of bromocresol green that is useful in the presentinvention is represented by Structure A.

The first ionization form of eosin Y that is useful in the presentinvention is represented by Structure B.

The first ionization form of erythrosin B that is useful in the presentinvention is represented by Structure C.

The first ionization form of pyrogallol red that is useful in thepresent invention is represented by Structure D.

The first ionization form of rosolic acid that is useful in the presentinvention is represented by Structure E.

The amines used to form the salt complex have structural characteristicswhich allow compatible interactions of the cation such that theresulting salt complex will be soluble in organic solvents, aqueoussolvents, and mixtures thereof. The amine is chosen to impart somelipophilicity to the salt complex which has some hydrophilic character.Where a user desires to increase the solubility of an organic reagent inan aqueous solvent, the user may wish to select an amine havingstructural characteristics which enhance ionization, polarization, orhydrogen bonding such as R groups which contain —OH, —SH, —C(O)NH₂,hydroxyalkyl, hydroxyalkoxyalkyl, and/or —CH₂CH₂O)_(n) functionality.Where a user desires to increase the solubility of an organic reagent inan organic solvent, the user may wish to select an amine having commonlyrecognized lipophilic features such as alkyl, alkenyl, alkynyl,haloalkyl, aromatic, or haloaromatic functional groups. The choice ofcounterion structure may also allow a user to inhibit precipitation orcrystallization through interaction of the R groups with the solvent.

The amine is the source of the cations for forming the salt complex uponpairing with the anions provided by the organic reagent source. Suitableamines for use in embodiments of the invention generally have thefollowing formula:H_(m)NR_(n.)The amine may be in a form having no net charge where m is 0, 1 or 2; nis 1, 2, or 3; and the sum of m and n is 3. The amine may also be in theammonium form, where the value of m is increased by 1. The amine mayfurther be in the charged quaternary form where n is 4 and m is equal tozero. R is an independently selected group, meaning that R may be thesame or different when n is greater than 1. The amines used in formingthe salt complex according to the present methods and products may havelipophilic properties, hydrophilic properties, or both lipophilic andhydrophilic properties.

Where a charged quaternary amine is used as the source of the cationsfor forming the salt complex, an ionized organic reagent such as anorganic acid reagent and a quaternary ammonium ion may be combined toform a salt complex where the ammonium ion is represented by the formulaNR₄ ⁺.R is an independently selected group, meaning that R may be the same ordifferent. The charged quaternary amines used in forming the saltcomplex according to the present methods and products may havelipophilic properties, hydrophilic properties, or both lipophilic andhydrophilic properties.

The ionized organic reagent and the quaternary ammonium ion may becombined through a variety of methods known in the art such as by an ionexchange process. For example, ion exchange can be performed using batchmode or column mode employing ion exchange media such as resin orcarbohydrate with ionizable groups. A typical ion exchange process foran organic acid may begin with loading the organic acid at an ionizingpH or as a salt, such as sodium, onto a prepared column of anion ionexchange media in a suitable form, such as hydroxide, to bind theorganic acid to the column. A solution of a quaternary ammonium saltsuch as tetraethylammonium chloride may then be applied to the column.As the chloride ion displaces the organic acid anion and releases theorganic acid anion from the column media, the remaining quaternaryammonium ion pairs with the ionized organic acid, allowing a solution ofthe quaternary ammonium salt of the organic acid to elute from thecolumn.

The desired degree of solubility of the salt complex may vary based uponthe type of solvent(s) required for the particular manufacturing processand the ultimate function of the final product. For example, aparticular protein dye may need to be soluble in an aqueous solvent(i.e., a solvent having a high water content) for one step of amanufacturing dip process but insoluble in the organic solvent of asubsequent step to inhibit the dye from washing out in the organicsolvent. For medical diagnostics, the dye salt generally needs topossess some aqueous solubility in order to be available for chemicalreactions with a biological sample such as urine. Further, the dye saltmay have functions such as a color change upon binding to protein thatcan be preserved or enhanced due to attraction to the lipophilic orhydrophilic portion of the protein. The amine that is used to form thesalt complex may impart these solubility and compatibility properties tothe dye.

The selection of a suitable R group(s) for use in the amine generallydepends on the desired degree of solubility of the amine in therespective organic solvent, aqueous solvent, or mixture thereof.Beginning with n=0 and m=3, amines generally have hydrophilic character.The R group(s) and the number of R group(s) may be selected to maintainthe hydrophilic character of the amine or to add lipophilic character tothe amine.

R is selected to provide sufficient lipophilicity or hydrophilicity tothe amine such that the resulting salt complex is soluble in the desiredorganic solvent, aqueous solvent, or mixture thereof and to allowcompatible interactions of the salt complex with the organic solvent,aqueous solvent, or mixture thereof. R is selected to render the saltcomplex soluble in aqueous solvents, organic solvents, and mixturesthereof. The desired solubility properties provided by R may be impliedfrom such data as octanol/water partition coefficients for suchsubstances as RH or ROH. In some embodiments, R may be a substituentgroup having both lipophilic and hydrophilic properties. In otherembodiments, at least one R group provides sufficient lipophilicity torender the salt complex soluble in an organic solvent and sufficienthydrophilicity to render the salt complex soluble in an aqueous solventor a mixture of an aqueous solvent and an organic solvent. R may beselected from, but is not limited to, alkyl, alkenyl, alkynyl,haloalkyl, aromatic, haloaromatic, alkylaminoacetyl, hydroxyalkyl, andhydroxyalkoxyalkyl substituent groups.

The selection of the value of m and n for use in the amine generallydepends upon the degree of hydrophilicity of the R group(s) which areselected and the reactivity of the amine toward other reagents. Forexample, where n=0, 1, or 2, the amine may participate in somenucleophilic reactions. If the user desires to increase the solubilityof the organic reagent to be applied to the diagnostic test device in anorganic solvent, the user may wish to select an amine with a hydrophobicsubstituent(s) having a higher value for n such as n=3 in trioctylamine.It is expected that the hydrophobic character of the salt complex willgenerally increase with increasing hydrophobic character of the Rgroup(s). For example, where R is changed from ethyl to butyl, thehydrophobic character of the salt complex will generally increasewhereas where R is changed from octyl to butyl, the hydrophobiccharacter of the salt complex will generally decrease.

Examples of suitable amines for use in embodiments of the inventioninclude, but are not limited to, trimethylamine, triethylamine, tributylamine, trioctylamine, tris(hydroxymethyl)aminomethane (TRIS),aminoethanol, butylamine, octylamine, triethanolamine, glucamine,polyethyleneglycolamines, amino acids, and mixtures thereof. In someembodiments, tris(hydroxymethyl)aminomethane (TRIS) is used. Althoughnot critical to its use, it is believed that the hydroxy groups in TRISare compatible with aqueous solvents and that the —CH₂OH groups alsopossess alkyl character which is compatible with organic solvents suchas methanol.

Where a charged quaternary amine is used in embodiments of theinvention, suitable quaternary ammonium salts include, but are notlimited to, tetrabutylammonium hydroxide, tetramethylammonium hydroxide,tetraethylammonium hydroxide, tetraproylammonium hydroxidemyristyltrimethylammonium bromide, cetyltrimethylammonium bromide,phenyltrimethylammonium bromide, tetrapentylammonium bromide,tetrahexylammonium bromide, tetraheptylammonium bromide,tetraoctylammonium bromide, and tetraoctadecylammonium bromide.

Examples of suitable quaternary ammonium ions for use in embodiments ofthe invention include, but are not limited to, tetrabutylammonium,tetramethylammonium, tetraethylammonium, tetraproylammoniummyristyltrimethylammonium, cetyltrimethylammonium,phenyltrimethylammonium, tetrapentylammonium, tetrahexylammonium,tetraheptylammonium, tetraoctylammonium, and tetraoctadecylammonium.

The ionic structures of some of the suitable amines for use inembodiments of the invention are shown as Structures F-J below. Atrioctylammonium ion that is useful in the present invention isrepresented by Structure F.

A triethylammonium ion that is useful in the present invention isrepresented by Structure G.

A triethanolammonium ion that is useful in the present invention isrepresented by Structure H.

An ammonium form of TRIS that is useful in the present invention isrepresented by Structure I.

A glucammonium ion that is useful in the present invention isrepresented by Structure J.

Once the salt complex is formed by the steps described above, the saltcomplex is dissolved in an organic solvent, an aqueous solvent, or amixture of one or more organic solvents and one or more aqueoussolvents. An example of a mixture of one or more organic solvents andone or more aqueous solvents is a mixture of an alcohol and water. Bydissolving the salt complex in an organic solvent, an aqueous solvent,or mixtures of one or more organic solvents and one or more aqueoussolvents, the reagent may be released into the solvent. The salt complexformed by mixing the organic reagent and the amine is generally solublein a wider range of solvents than the unsalted organic reagent would bealone.

Examples of suitable organic solvents in which the salt complex may bedissolved include, but are not limited to, alcohols, tetrahydrofuran(THF), and mixtures of toluene and THF. Examples of suitable aqueoussolvents in which the salt complex may be dissolved include, but are notlimited to, water and mixtures of alcohol and water. Examples ofsuitable solvents for a multiple-dip application process such as atwo-dip application include, but are not limited to, an alcohol-watermixture and a THF-toluene mixture.

Upon dissolving the salt complex in the desired solvent, the reagent isreleased into the solvent. When a user then dips an absorbent materialsuch as paper into the desired solvent, the reagent that is present inthe solvent becomes absorbed into the absorbent material. Users cangenerally control the degree of reagent solubility in the solvent ortest sample by selecting a particular amine and a particular organicreagent such that the resulting salt complex has the desired degree ofsolubility in organic solvents, aqueous solvents, and mixtures thereof.The methods described herein and the diagnostic test devices prepared bythe methods described herein allow users to generally control the degreeof reagent solubility in the various dip solutions of a multiple dipapplication process.

Once the reagent has been absorbed into the absorbent component by themethods described above, the absorbent component is dried and assembledinto a diagnostic test device such as a diagnostic test strip for use indetecting the presence of an analyte(s) in organic test samples, aqueoustest samples, and sample mixtures of one or more organic solvents andone or more aqueous solvents. Examples of suitable test samples includebody fluids and diluted aqueous mixtures thereof.

The following examples are given to exemplify embodiments of theinvention. These examples should not be construed to limit the inventionas otherwise described and claimed herein.

EXAMPLE 1

A first aqueous solution was formed by the addition of three submixes(Submixes 1-3). To form Submix 1, 10 ml of methanol, 1.76 ml of 125 mMtris(hydroxymethyl)aminomethane (TRIS) in methanol and 44.0 mg ofpyrogallol red were added together. These components were mixed forabout 20 to about 30 minutes to form Submix 1. To form Submix 2, 4.10 g(3.20 mL) of 40% phytic acid, 8.00 mL of 5% aqueous PVA (polyvinylalcohol) of 31-50K, 11.2 mL of 1N NaOH, 0.654 mL of 100 mg/mL of aqueoussodium molybdate, and 45.6 mL of water were added together. The NaOH wasadded to adjust the pH to approximately 2.3. These components were mixedfor about 60 minutes to form Submix 2. To form Submix 3, 217.5 mg ofdisodium oxalate, 120.0 mL of 500 mM L-citrulline with a pH 2.5 and100.0 mg of Cibacron Brilliant Yellow were added together. Thecomponents were mixed for about 30 minutes to form Submix 3.

Submix 1 and Submix 2 were mixed together for about 10 minutes. Next,Submix 3 was added to the combination of Submix 1 and Submix 2 and wasmixed until uniform. The mixture of Submixes 1, 2, and 3 was adjusted topH 2.6. Submixes 1-3 were used to form a First Dip solution adjustedwith water to a volume of 200 mL.

The reagent paper was dipped into the First Dip solution of 200 mL anddried in a three-stage tunnel oven at 50/50/70° C. with a 1 inch airflow. The reagent paper used was made from 4 inch Ahlstrom 204 paper.

The Second Dip solution was made by the following procedure. 0.48 g ofKOK (a polypropyleneglycol carbonate polymer) such as disclosed in U.S.Pat. No. 5,424,215 was weighed directly into a beaker. Then, 36.57 mg ofDNHB (5,5″ Dinitro -3′,3″,3,4,5,6-hexabromophenolsulfonephthalein) wasadded to the container containing KOK. 16 mL of stabilized THF was addedto the mixture of KOK and DNHB in a fume hood. 144 mL of toluene wasadded to the mixture of KOK, DNHB, and THF and mixed until homogenous toform the Second Dip solution. The Second Dip solution was stable for 24hours.

The reagent paper was then dipped into the Second Dip solution of 160mL. The diagnostic reagent paper was dried in a three-stage tunnel ovenat 50/50/70° C. with a 1 inch air flow. The paper was cut to form theactive pad of the diagnostic test strips. The diagnostic strips includedthe active pad that was adhered to a polymeric strip.

EXAMPLE 2

Five 5 mM solutions of the dyes listed in Tables 1-5 (i.e., bromocresolgreen, eosin Y, rosolic acid, 8-anilino-1-sulfonic acid, erythrosin B)in 96% methanol, 2% ethanol and 2% water were prepared. The five dyesolutions were then treated with 1 equivalent of each base listed inTables 1-5 (i.e., sodium hydroxide, TRIS, and triethylamine) to formsalt solutions (i.e., sodium salt solutions, TRIS salt solutions, andtriethylamine salt solutions respectively).

A 50 μL portion of each salt solution was then mixed with 950 μL waterand 1000 μL butanol. Extractions were then performed in Mixxorextractors available from Aldrich Chemical Company. Where necessary todeal with lingering interface phases, the samples were centrifuged.

Tests were then performed on the respective salt solutions to determinewhether the salts of the respective dyes dissolved preferentially inbutanol (a more lipophilic solvent) or water (a less lipophilicsolvent). The ratio of the dye concentration for the butanol layerversus the water layer was determined by spectroscopy using a HewlettPackard 8453 diode array spectrophotometer and a 1 mL quartz cuvette.Spectroscopy versus the butanol blank or the water blank was done as 50μL upper (butanol) or lower (aqueous) phase plus 950 μL correspondingsolvent. All absorbances were blanked on solvent in a quartz cuvette andwere baseline corrected at an absorbance of 900 nm before subsequentcalculations to correct for cuvette placement, dust, etc. Allabsorbances were then measured at the wavelengths noted below.

Before determining the butanol to water absorbance levels, theabsorbance of the solute in butanol was corrected for solvent dependentshifts in wavelength maximum and intensity based on the referencespectra of known gravimetric quantities of the dye salts in butanol andwater. For such cases, the partition ratio of butanol absorbance towater absorbance was calculated using the following formula:Partition Ratio=Sample Absorbance in Butanol×Intensity Factor/SampleAbsorbance in Waterwhere the intensity factor was calculated using the following formula:Intensity Factor=Reference Sample Absorbance at λ_(max) inwater/Reference Sample Absorbance at λ_(max) in butanolThe intensity factor adjusts for those cases where the sameconcentration of dye in the two different solvents produced twodifferent absorbances.

The water absorbances, butanol absorbances, intensity factors, rawabsorbance ratios, and partition ratios of the respective salt solutionsare shown below in Tables 1-5.

TABLE 1 Butanol/Water Partitioning of Bromocresol Green Salts Raw Ratioof Partition Ratio Butanol of Butanol Absorbance Absorbance IntensityAbsorbance to Absorbance to of Base in of Base in Factor Water WaterBase Water¹ Butanol² (IF) Absorbance Absorbance Sodium Hydroxide 0.1750.315 1.03 1.8 1.9 TRIS 0.146 0.250 2.47 1.7 4.2 Triethylamine 0.0970.237 2.58 2.4 6.3 ¹The wavelength (λ_(max)) was 615 nm. ²The wavelength(λ_(max)) was 630 nm.

TABLE 2 Butanol/Water Partitioning of Eosin Y Salts Raw Ratio ofPartition Ratio Butanol of Butanol Absorbance Absorbance of IntensityAbsorbance to Absorbance to of Base in Base in Factor Water Water BaseWater³ Butanol⁴ (IF) Absorbance Absorbance Sodium Hydroxide 0.700 0.1281.04 0.18 0.19 TRIS 0.703 0.174 1.00 0.25 0.25 Triethylamine 0.705 0.1870.99 0.26 0.26 ³The wavelength (λ_(max)) was 518 nm. ⁴The wavelength(λ_(max)) was 530 nm.

TABLE 3 Butanol/Water Partitioning of Rosolic Acid Salts Raw Ratio ofPartition Ratio Butanol of Butanol Absorbance Absorbance of IntensityAbsorbance to Absorbance to of Base in Base in Factor Water Water BaseWater^(5,6) Butanol⁷ (IF) Absorbance Absorbance Sodium Hydroxide 0.0850.147 0.69 1.7 1.2 TRIS 0.003 0.055 1.07 17 18 Triethylamine 0.006 0.0770.94 12 11 ⁵The wavelength (λ_(max)) was 547 nm. ⁶Due to the strongsolvent dependent spectral intensity and predominant butanolpartitioning of the rosolic acid salt, the aqueous solvent for thereference sample of rosolic acid salt was saturated with butanol beforemeasuring its absorbance. The absorbance of the aqueous layer in thecase of the rosolic acid salt was very weak so a solution that was fourtimes less dilute was measured. Thus, the aqueous absorbances shown inTable 3 above have been divided by four. ⁷The wavelength (λ_(max)) was562 nm.

TABLE 4 Butanol/Water Partitioning of 8-Anilino-1-Sulfonic Acid SaltsRaw Ratio of Partition Ratio Butanol of Butanol Absorbance of Absorbanceof Intensity Absorbance to Absorbance to Base in Base in Factor WaterWater Base Water⁸ Butanol⁹ (IF) Absorbance Absorbance Sodium Hydroxide0.065 0.121 0.98 1.9 1.8 TRIS 0.050 0.129 0.89 2.6 2.3 Triethylamine0.045 0.156 1.01 3.4 3.5 ⁸The wavelength (λ_(max)) was 265 nm. ⁹Thewavelength (λ_(max)) was 269 nm.

TABLE 5 Butanol/Water Partitioning of Erythrosin Salts Raw Ratio ofPartition Ratio Butanol of Butanol Absorbance of Absorbance of IntensityAbsorbance to Absorbance to Base in Base in Factor Water Water BaseWater¹⁰ Butanol¹¹ (IF) Absorbance Absorbance Sodium Hydroxide 0.5740.237 0.93 0.41 0.38 TRIS 0.631 0.196 0.90 0.31 0.28 Triethylamine 0.5960.240 1.04 0.40 0.42 ¹⁰The wavelength (λ_(max)) was 528 nm. ¹¹Thewavelength (λ_(max)) was 536 nm.

The data set forth in Tables 1-5 above shows that higher ratios ofpartitioning of the respective dye salts in the butanol layer versus thewater layer were obtained with triethylamine, the most lipophilic of theamines tested. These results indicate that the salt became morelipophilic in the presence of the triethylammonium cation, enabling moreof the salt to dissolve in the butanol layer, the more lipophilicsolvent.

EXAMPLE 3

A solution of pyrogallol red at 2 mg/mL in methanol with 2% ethanol and2% water was prepared. The pyrogallol red solution was then treated withone equivalent of each base listed in Table 6 (i.e., triethanolamine,glucamine, sodium hydroxide, trioctylamine, TRIS, and triethylamine) toform the respective pyrogallol red salt solutions (i.e., triethanolaminesalt solution, glucamine salt solution, sodium salt solution,trioctylamine salt solution, TRIS salt solution, and triethylamine saltsolution respectively).

Aliquots of 50 μL of each pyrogallol red salt solution were then mixedwith 950 μL water and 1000 μL butanol. Extractions were then performedin Mixxor extractors.

Tests were then performed on the respective salt solutions to determinewhether the salts of the pyrogallol red dye dissolved preferentially inbutanol (a more lipophilic solvent) or water (a less lipophilicsolvent). The ratio of the dye concentration for the butanol layerversus the water layer was determined by spectroscopy using a HewlettPackard 8453 diode array spectrophotometer and a 1 mL quartz cuvette.Spectroscopy versus the butanol blank or the water blank was done as 50μL upper (butanol) or lower (aqueous) phase plus 950 μL correspondingsolvent. All absorbances were blanked on solvent in a quartz cuvette andwere baseline corrected at an absorbance of 900 nm before subsequentcalculations to correct for cuvette placement, dust, etc. and were thenmeasured at 276 nm.

TABLE 6 Butanol/Water Partitioning of Pyrogallol Red Dye Salts ofVarious Bases Absorbance Absorbance Ratio of Butanol of Dye Salt of DyeSalt Absorbance to Water Base in Butanol in Water AbsorbanceTriethanolamine 0.044 0.296 0.15 Glucamine 0.030 0.328 0.09 SodiumHydroxide 0.034 0.338 0.10 Trioctylamine 0.155 0.049 3.17 TRIS 0.0410.326 0.13 Triethylamine 0.063 0.274 0.23

The data set forth in Table 6 above shows that higher ratios ofpartitioning of the respective dye salts in the butanol layer versus thewater layer were obtained with trioctylamine, the most lipophilic of theamines tested. These results indicate that the salts became morelipophilic in the presence of the trioctylammonium cation, enabling moreof the salt to dissolve in the butanol layer, the more lipophilicsolvent.

EXAMPLE 4

A solution of pyrogallol red at 2 mg/mL in methanol with 2% ethanol and2% water was prepared. The pyrogallol red solution was then treated withone equivalent of the following amines: TRIS, triethanolamine,glucamine, triethylamine, trioctylamine, and sodium to form therespective pyrogallol red salt solutions listed in Table 7 (i.e., TRISsalt solution, triethanolamine salt solution, glucamine salt solution,triethylamine salt solution, trioctylamine salt solution, and sodiumsalt solution respectively).

Aliquots of 100 μL of each pyrogallol red salt solution were then mixedwith solutions of 0 to 100 μL toluene and methanol for the remainder tomake 1000 μL solutions. Toluene is a non-polar, organic solvent which isgenerally lipophilic. The 1000 μL solutions were allowed to formprecipitate by standing overnight in sealed vials. The samples were thencentrifuged.

The fluid portions of the centrifuged samples were then analyzed byspectroscopy. An analysis of the ratio of the concentration of therespective salt solutions in toluene was measured against a methanolblank. This analysis was performed to determine the solubility thresholdof pyrogallol red salts in solvent having an increasing percentage oftoluene. The solubility of the fluid portions of the centrifuged sampleswas determined spectroscopically using a Hewlett Packard 8453 diodearray spectrophotometer and a 1 mL quartz cuvette. Spectroscopy versusthe methanol blank was conducted as 50 μL fluid plus 950 μL methanol.The percent recovery of the spin supernates (i.e., how much of thepyrogallol red salt remained in solution) was determined at anabsorbance of 518 nm and was compared to the absorbance level of asolution containing no toluene, which provided complete and stablesolubility (100% recovery) of all of the salts.

TABLE 7 Stability of Pyrogallol Red in Solvents of IncreasingLipophilicity as Percent Recovery of Absorbance Percent TriethanolamineGlucamine Triethylamine Trioctylamine Toluene TRIS Salt Salt Salt SaltSalt Sodium Salt 0

20

40

60

70

75

58

80

44

 68 83

85  46  23

61 86

87  20  19  15

88  36

89  1  23  7

90  16  7  5

4 * The shaded entries in Table 7 above represent the proportions oftoluene in solvent where each salt remained soluble overnight.

The data set forth in Table 7 above shows the stability of pyrogallolred in solvents having increasing lipophilicity represented as thepercent recovery of solution absorbance. The data set forth in Table 7above shows that the more lipophilic cations such as those from TRIS,triethylamine, and trioctylamine enabled more of the pyrogallol red dyesalt to remain in a solution having a high content of a non-polar,organic component such as toluene.

EXAMPLE 5

An automated protein assay was devised for the BioTek, Precision 200096/384 Well Microplate Automated Pipetting System to determine therelative detection of different proteins using salts of pyrogallol red.The dilutor was programmed for reactions of 180 μL reagent+40 μL sample.The reagent was 250 mM citrulline buffer (at pH 2.5) with 160 μMmolybdate and 54 μM pyrogallol red salt. The samples were phosphatebuffered saline (blank), human serum albumin (HSA) 25 mg/dL, and otherproteins (i.e., human IgG, Tammm-Horsfall, and Lambda Light Chain) at 25mg/dL.

Absorbance rate data were collected on a Biotek, Powerwave X MicroplateAbsorbance reader using BioTek “KC Jr” software to determine therelative detection of the different proteins (i.e., HSA, human IgG,Tammm-Horsfall, and Lambda Light Chain) using salts of pyrogallol red.Two sets of replicates of the different proteins were run. Theabsorbance was measured at 600 nm (the absorbance frequency of bluecolors), and the baseline absorbance at 900 nm (an absorbance frequencybeyond the visible range) was subtracted after 10 minutes at 37°Centigrade. The protein response was determined by subtracting thephosphate buffered saline sample reaction result from the proteinreaction result. The net absorbance at 600 nm and at 900 nm at 10minutes reaction time is shown below in Table 8.

TABLE 8 Reaction Absorbances at 10 Minutes Reaction Time SodiumTrioctylamine Pyrogallol Red Pyrogallol Red Salt in Sodium Salt inTrioctylamine Phosphate Pyrogallol Phosphate Pyrogallol Red BufferedSaline Red Salt Buffered Saline Salt with Protein (No Protein) withProtein (No Protein) Protein 600 nm Absorbance Levels REPLICATE #1 HSA0.197 0.211 0.476 0.454 IgG 0.199 0.207 0.482 0.475 Tamm- 0.196 0.2030.485 0.454 Horsfall Lambda 0.198 0.204 0.485 0.446 Light ChainREPLICATE #2 HSA 0.196 0.211 0.483 0.464 IgG 0.200 0.208 0.489 0.474Tamm- 0.198 0.204 0.486 0.45 Horsfall Lambda 0.196 0.201 0.485 0.44Light Chain 900 nm Absorbance Background Levels REPLICATE #1 HSA 0.0350.035 0.082 0.087 IgG 0.036 0.037 0.081 0.092 Tamm- 0.036 0.036 0.0810.085 Horsfall Lambda 0.034 0.035 0.081 0.086 Light Chain REPLICATE #2HSA 0.035 0.035 0.081 0.088 IgG 0.036 0.036 0.082 0.093 Tamm- 0.0350.036 0.082 0.084 Horsfal Lambda 0.035 0.036 0.08 0.087 Light Chain

The net protein response for trioctylamine versus sodium salts ofpyrogallol red for the protein samples minus the non-protein samples areshown in Table 9 below. The absorbance at 600 nm at 10 minutes reactiontime was corrected by the baseline absorbance at 900 nm and is shownbelow in Table 9 as averaged data.

TABLE 9 Detection of Proteins by Two Pyrogallol Red Salts Sodium SaltTrioctylamine Salt Reaction Absorbance at Reaction Absorbance at 600 nm− Absorbance 600 nm − Absorbance at Protein at 900 nm 900 nm HSA 0.015−0.021 IgG 0.008 −0.011 Tamm-Horsfal 0.007 −0.034 Lambda Light Chain0.006 −0.042

This experiment showed a modest response to protein from the sodium saltof pyrogallol red. The absorbance at 600 nm for the protein samplesranged from 0.201 to 0.211 while the absorbance at 600 nm for thenon-protein sample (i.e., the PBS blanks) was lower, ranging from 0.196to 0.200. Following the 900 nm absorbance background subtraction, theaverage difference between the protein and non-protein reactions rangedfrom 6 to 15 milli absorbance units.

However, the highly lipophilic cation, trioctylammonium, caused animmediate, strong blue color of the pyrogallol red reagent solution inthe non-protein blank which was indistinguishable in color from theprotein reaction. The absorbance levels of the non-protein blanks wereslightly stronger than the absorbance levels of the protein reactions.The blue color of the trioctylammonium pyrogallol red did not dissipatewhen incubated in the citrulline buffer whether with protein (absorbanceat 600 nm=0.44 to 0.48) or without protein (absorbance at 600 nm=0.48 to0.49). This data suggests that the ion pairs of anionic dye andlipophilic cation may remain associated and stable over moderate periodsof time even in the presence of large amounts of sodium cation.

EXAMPLE 6

An automated protein assay was devised for the BioTek, Precision 200096/384 Well Microplate Automated Pipetting System to determine therelative detection of different proteins using salts of pyrogallol red.The dilutor was programmed for reactions of 180 μL reagent+40 μL sample.The reagent was 250 mM citrulline buffer (at pH 2.5) with 160 μMmolybdate and 74 μM pyrogallol red salt. The samples were phosphatebuffered saline (blank), human serum albumin (HSA) 25 mg/dL, and otherproteins (i.e., human IgG, Tammm-Horsfall, and Lambda Light Chain) at 25mg/dL.

Absorbance data were collected on a Biotek, Powerwave X MicroplateAbsorbance reader using BioTek “KC Jr” software to determine therelative detection of the different proteins (i.e., HSA, human IgG,Tammm-Horsfall, and Lambda Light Chain) using salts of pyrogallol red.Two sets of replicates of the different proteins were run. Theabsorbance was measured at 600 nm and the baseline absorbance at 900 nmwas subtracted after 10 minutes at 37° Centigrade. The protein responsewas determined by subtracting the phosphate buffered saline samplereaction result from the protein reaction result. The average netabsorbance for the two sets of replicates at 600 nm at 10 minutesreaction time after subtracting the phosphate buffered saline (PBS)sample reaction result is shown below in Table 10.

TABLE 10 Detection of Different Proteins by Five Pyrogallol Red SaltsSERIES #1 Sodium Triethylamine Pyrogallol TRIS Pyrogallol Red PyrogallolRed Protein Red Salt Salt Salt HSA 0.0536 0.0750 0.0600 IgG 0.03110.0410 0.0285 Tamm-Horsfall 0.0141 0.0175 0.0135 Lambda Light Chain0.0041 0.0030 0.0020 SERIES #2 Sodium Triethylamine Pyrogallol GlucaminePyrogallol Pyrogallol Red Protein Red Salt Red Salt Salt HSA 0.05630.0618 0.0559 IgG 0.0318 0.0318 0.0284 Tamm-Horsfall 0.0118 0.01230.0109 Lambda Light Chain 0.0037 0.0088 0.0049The results for protein equivalence of the non-albumin proteins wereexpressed as a percentage of the protein colorimetric response to HSA asshown in Table 11 below.

TABLE 11 Percent Detection of Different Proteins by Five Pyrogallol RedSalts as Percentage of HSA Detection SERIES #1 Sodium TriethylaminePyrogallol TRIS Pyrogallol Red Pyrogallol Red Protein Red Salt Salt SaltIgG 58 55 48 Tamm-Horsfall 26 23 22 Lambda Light Chain 8 4 3 SERIES #2Sodium Triethylamine Pyrogallol Glucamine Pyrogallol Pyrogallol RedProtein Red Salt Red Salt Salt IgG 56 51 51 Tamm-Horsfall 21 20 19Lambda Light Chain 7 14 9

As shown in Table 11 above, compared to sodium salt, the four salts withmore complex cations gave roughly similar results for the IgG andTamm-Horsfall proteins. The more lipophilic triethylammonium counterionwas less responsive (3%) than sodium (8%) to the Lambda Light Chainprotein. The more hydrophilic glucammonium cation was more responsive(14%) than sodium (8%) to the Lambda Light Chain protein. The sodiumsalt of pyrogallol red is known to exhibit different affinity forbinding and, therefore, a different detection response to differentproteins. Example 6 demonstrates that the relative affinity of ionizeddyes for different proteins is affected by the nature of theircounterions.

While the invention has been described with a number of embodiments, thescope of the invention is not intended to be limited by the specificembodiments. Modifications and variations from the described embodimentsexist. For example, while the invention is described in connection withtest samples comprising biological fluids such as human urine, suitabletest samples may also include agricultural or environmental fluids.

In addition, although the inventive methods and products have beendescribed in connection with organic acid reagents such as acidic dyes,it is contemplated that the technique may also be applied to anorganic-soluble enzyme substrate such as bromo, chloroindolyl phosphate.It is contemplated that an organic-soluble enzyme substrate can also becombined with an amine having the hydrophilic properties discussed abovesuch that an enzyme substrate which is soluble in organic solvents buthas no solubility or only limited solubility in aqueous solvents can berendered soluble in aqueous solvents.

Also, although the inventive methods and products have been described inconnection with reagents which are organic acids, it is contemplatedthat the technique may also be applied to an organic molecule which hasgroups capable of developing a positive charge, such as an organicamine. For example, an organic amine could be countered with RSO₃H orROPO₃H or RCO₂H where R is selected based upon the desired hydrophilicor hydrophobic properties in a similar manner as described above.

While particular embodiments and applications of the present inventionhave been illustrated and described, it is to be understood that theinvention is not limited to the precise construction and compositionsdisclosed herein and that various modifications, changes, and variationsmay be apparent from the foregoing descriptions without departing fromthe spirit and scope of the invention as defined in the appended claims.

1. A method of applying an organic aromatic acid dye other thanpyrogallol red having limited solubility in aqueous solvents to acomponent of a diagnostic test device comprising: mixing said organicaromatic acid dye other than pyrogallol red and tris(hydroxyl methyl)amino methane to form a salt complex in solution, said organic aromaticacid dye other than pyrogallol red having at least one functional groupselected from a carboxylic acid, a sulfonic acid, a phosphoric acid anda mixture thereof; adding the salt complex in solution to a solvent torelease the salt complex into the solvent; and applying the solventcontaining said salt complex to the component of the diagnostic testdevice such that said salt complex present in the solvent becomesintegrated into the diagnostic test device.
 2. The method of claim 1,wherein said organic aromatic acid dye is a phenolsulfonephthalein. 3.The method of claim 1, wherein the diagnostic test device is adiagnostic test strip and the component is paper.
 4. The method of claim3, wherein the diagnostic test strip is adapted to detect the presenceof an analyte in a fluid.
 5. The method of claim 4, wherein the analyteis a protein.
 6. A method of applying an organic aromatic acid dye otherthan pyrogallol red having limited solubility in aqueous solvents to acomponent of a diagnostic test device comprising: mixing said organicaromatic acid dye other than pyrogallol red and an amine to form a saltcomplex in solution, the amine is represented by the formulaHmNRn wherein m is 0, 1, or 2; n is 1, 2, or 3; the sum of m and n is 3;and R is an independently selected organic group which renders the saltcomplex more soluble in aqueous solvents, said organic aromatic acid dyeother than pyrogallol red being a phenolsulfonephthalein; adding thesalt complex in solution to a solvent to release the salt complex intothe solvent; and applying the solvent containing said salt complex tothe component of the diagnostic test device such that said salt complexpresent in the solvent becomes integrated into the diagnostic testdevice.
 7. The method of claim 6, wherein the amine istris(hydroxymethyl)aminomethane.
 8. A method of applying an organicaromatic acid dye other than pyrogallol red having limited solubility inaqueous solvents to a component of a diagnostic test device comprising:mixing an organic aromatic acid dye other than pyrogallol red having atleast one functional group selected from a carboxylic acid, a sulfonicacid, a phosphoric acid and a mixture thereof and tris (hydroxylmethyl)methane to form a salt complex in solution; adding the salt complex insolution to a solvent to release said salt complex into the solvent; andapplying the solvent containing said salt complex to the component ofthe diagnostic test device such that said salt complex present in thesolvent becomes integrated into the diagnostic test device.
 9. Themethod of claim 8, wherein said organic aromatic acid dye is aphenolsulfonephthalein.
 10. A method of applying an organic aromaticacid dye other than pyrogallol red having limited solubility in aqueoussolvents to a component of a diagnostic test device comprising: mixing aphenolsulfonephthalein and an amine selected from trimethylamine,triethylamine, tributyl amine, trioctylamine, tris (hydroxymethyl)aminomethane, aminoethanol, butylamine, octylamine, triethanolamine,glucamine, a polyethyleneglycolamine, an amino acid, and mixturesthereof to form a salt complex in solution; adding the salt complex insolution to a solvent to release said salt complex into the solvent; andapplying the solvent containing said salt complex to the component ofthe diagnostic test device such that said salt complex present in thesolvent becomes integrated into the diagnostic test device.
 11. Themethod of claim 10, wherein the amine istris(hydroxymethyl)aminomethane.
 12. The method of claim 10, wherein thediagnostic test device is adapted to detect the presence of a protein ina fluid.
 13. The method of claim 10, wherein the diagnostic test deviceis a diagnostic test strip and the component is paper.
 14. A method ofapplying an organic aromatic acid dye other than pyrogallol red havinglimited solubility in aqueous solvents to a paper of a diagnostic teststrip comprising: mixing tris(hydroxymethyl)aminomethane and an organicaromatic acid dye other than pyrogallol red having an acid functionalgroup selected from a carboxylic acid, a sulfonic acid, a phosphoricacid, and mixtures thereof to form a salt complex in solution; addingthe salt complex in solution to a solvent to release said salt complexinto the solvent; and applying the solvent containing said salt complexto the paper of the diagnostic test strip such that said salt complexpresent in the solvent becomes integrated into the paper.
 15. The methodof claim 14, wherein the organic aromatic acid dye is aphenolsulfonephthalein.
 16. A method of applying an organic aromaticacid dye other than pyrogallol red having limited solubility in aqueoussolvents to a paper of a diagnostic test strip comprising: mixing anamine selected from trimethylamine, triethylamine, tributyl amine,trioctylamine, tris(hydroxymethyl)aminomethane, aminoethanol,butylamine, octylamine, triethanolamine, glucamine, apolyethyleneglycolamine, an amino acid, and mixtures thereof and aphenolsulfonephthalein to form a salt complex in solution; adding thesalt complex in solution to a solvent to release said salt complex intothe solvent; and applying the solvent containing said salt complex tothe paper of the diagnostic test strip such that said salt complexpresent in the solvent becomes integrated into the paper.
 17. The methodof claim 16, wherein the amine is tris(hydroxymethyl)aminomethane.
 18. Adiagnostic test device containing an organic aromatic acid dye otherthan pyrogallol red having limited solubility in aqueous solventsprepared by the process of mixing an organic aromatic acid dye otherthan pyrogallol red and tris(hydroxymethyl)aminomethane, said organicaromatic acid dye having at least one functional group selected from acarboxylic acid, a sulfonic acid, a phosphoric acid and a mixturethereof; adding the salt complex in solution to a solvent to releasesaid complex into the solvent; and applying the solvent containing saidsalt complex to a component of the diagnostic test device such that saidsalt complex present in the solvent becomes integrated into thediagnostic test device.
 19. The diagnostic test device of claim 18,wherein the organic aromatic acid dye is a phenolsulfonephthalein. 20.The diagnostic test device of claim 18, wherein the diagnostic testdevice is adapted to detect the presence of an analyte in a fluid. 21.The diagnostic test device of claim 20, wherein the analyte is aprotein.
 22. A diagnostic test device containing an organic aromaticacid dye other than pyrogallol red having limited solubility in aqueoussolvents prepared by the process of mixing an organic aromatic acid dyeother than pyrogallol red and an amine to form a salt complex insolution, the amine is represented by the formulaHmNRn wherein m is 0, 1, or 2; n is 1, 2, or 3; the sum of m and n is 3;and R is an independently selected organic group which renders the saltcomplex more soluble in aqueous solvents, said organic aromatic acid dyebeing a phenolsulfonephthalein; adding the salt complex in solution to asolvent to release said complex into the solvent; and applying thesolvent containing said salt complex to a component of the diagnostictest device such that said salt complex present in the solvent becomesintegrated into the diagnostic test device.
 23. The diagnostic testdevice of claim 22 wherein the amine is tris(hydroxymethyl)aminomethane.24. The diagnostic test device of claim 22 wherein the diagnostic testdevice is a diagnostic test strip and the component is paper.